IgA nephropathy (IgAN) is the most common glomerulonephritis throughout the world. The disease is characterized by the deposition of immunoglobulin A1 (IgA1) in the glomerulus. IgAN is best explained by a four hit model, where each hit must occur for the disease to progress. Hit 1, the focus of this proposal, entails the production of galactose (Gal)-deficent IgA1. The second hit is the production of anti-Gal-deficient IgA1 autoantibodies. In hit 3, autoantibodies bind Gal-deficient IgA1 in the serum of patients to form circulating immune complexes. Hit 4 is comprised of the localization of the circulating immune complexes to the mesangium of the glomeruli where they lead to mesangial cell proliferation, inflammation, and scarring and eventually end stage renal disease. This proposal focuses on a key step in the first hit, where IgA1 glycosylation is initiated by a family f enzymes known as GalNAc- transferases (GalNAc-T). The main objective of the project is to characterize the role of GalNAc-Ts in the disease IgAN. To do this, we will employ novel strategies using high-resolution mass spectrometry to first characterize the native glycosylation of IgA1 by the enzyme GalNAc-T2. This will be feasible due to the glycoform specific resolution of reaction products that can be obtained with mass spectrometry analysis. GalNAc- T2 has previously been shown to be important in IgA1 hinge region (HR) glycosylation. The hinge region of IgA1 contains two overlapping decapeptide repeats and 9 potenial sites of glycosylation. We will characterize how GalNAc-T2 glycosylates IgA1 HR in a time dependent manner while also investigating the roles of the two primary domains of GalNAc-T2: the lectin domain and the catalytic domain (aims 1 and 2). After establishing a baseline for glycosylation initiation of IgA1 HR by GalNAc- T2, we will explore the role of another GalNAc-T family member GalNAc-T14 in the disease IgAN (aim 3). We have previously identified GalNAc-T14 to be important in IgAN, but using the methods proposed in conjunction with the data on GalNAc-T2 native glycosylation, we will demonstrate isozyme specific involvement of GalNac-T14 in IgAN. In addition, we will identify the subdomains responsible for disease associated changes in glycosylation, revealing novel and specific potential drug targets. Successful completion of these aims will provide a fundamental understanding of the biochemistry that result in a key hit in the IgAN pathogenicity model. This will open the door to new therapeutic opportunities. Furthermore the strategies developed will be useful in a variety of other disease associated with altered glycosylation.